Pneumococci are major pathogens that continue to cause invasive disease in humans. Acquisition of resistance to many of the most useful antibacterial agents including penicillin has been reported and such resistant pneumococci have already caused human disease causing serious problems to chemotherapy, including treatment failure. Any strategy for the prevention of the spread of these bacteria requires a better understanding of the nature of genetic determinants, their possible origin and mode of dissemination and the biochemical basis of resistance. Our previous studies have established that penicillin resistance mutations cause remodeling of the pneumococcal penicillin binding proteins (PBPs) as evidenced by changes in drug affinity, copy number, and electrophoretic pattern of one or the other of four PBPs. An unexpected finding was that several of the highly penicillin resistant clinical isolates was also tolerant to penicillin: even when treated with the elevated concentrations of penicillin above the minimum growth inhibitory concentration (MIC) value, these cells would not lyse and would lose viability only slowly. Preliminary data suggest an alteration in autolysin control. The program planned for this application will have four points of concentration. 1) We plan to better define the number of genetic loci of penicillin resistance using genetic transformation with DNA isolated from clinical isolates from various geographic locales, various resistance levels and PBP patterns, and heterologous DNA from penicillin resistance STreptococcus viridans. We plan to clone the pneumococcal binding proteins, using homologous or heterologous cloning vehicles. 2) Biochemical experiments are planned to try to resolve the PBP alterations on the polypeptide level, using partial proteolytic digestion coupled with two-dimensional electrophoretic separation of acylated (penicilloyl) peptides from penicillin sensitive and isogenic resistant pneumococci, constructed by transformation. 3) The composition and fine structure of cell walls from penicillin sensitive and isogenic resistant transformants will be studied, using a combination of affinity chromatography and high pressure liquid chromatography (HPLC) systems. 4) The biochemical basis of the newly discovered penicillin tolerance trait and the mechanism by which penicillin triggers autolytic activity will be investigated.